The cystic fibrosis (CF) lung is colonized by a polymicrobial community, and pulmonary disease resulting from this chronic infection is the leading cause of morbidity and mortality in CF patients. CF lung infections are notoriously difficult to treat and current antibiotic therapies do not clear these infections. Culture-independent approaches (i.e., deep sequencing) characterizing CF lung communities has revealed that there are many more bacteria capable of colonizing the CF lung than previously recognized by conventional culturing techniques. For most bacteria identified in deep sequencing studies, the physiology and pathogenesis in the context of the CF lung are unknown. To date, only a handful of CF pathogens have been investigated for their responses to clinically relevant antibiotics and for their growth, biofilm formation and cytotoxicity kinetic. Our central hypothesis is that polymicrobial interactions in the CF lung play important roles in antibiotic tolerance and that understanding the mechanisms of these interactions will inform future therapeutic developments. Using our in vitro co-culture model of bacterial biofilm formation on CF-derived airway cells, we have begun to characterize these bacteria in single and mixed communities. With this approach, we have discovered a mixed species induced hypersensitivity of Pseudomonas aeruginosa to Cayston, a commonly prescribed CF maintenance therapy antibiotic, when co- cultured in the presence of Streptococcus salivarius. Here, we will test the hypothesis that recapitulating polymicrobial communities in our in vitro co-culture model of bacterial biofilm formation will elucidate additional interspecies interactions an determine the molecular mechanisms underlying the contributions of polymicrobial interactions to antibiotic tolerance. !